The emergence of in vivo resistance of Plasmodium falciparum malaria to chloroquine and to the combination antifolate drug pyrimethamine-sulfadoxine (PS) is a major threat to public health in Africa. The overall aim of this project is to define the mechanisms of resistance to these drugs through a combination of field studies in Malawi (linked to independently funded projects at other African sites) and laboratory studies at the University of Maryland. Point mutations in parasite dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) confer in vitro resistance to pyrimethamine and sulfadoxine, respectively. Evidence is now emerging that specific genotypes of a newly described gene, candidate gene 2 (cg2), are similarly responsible for in vitro chloroquine resistance, although other genes may also be involved. The relationships between these mutations and genotypes and the clinical outcomes of treatment with PS and chloroquine remain poorly understood, and reliable molecular methods for surveillance for drug-resistant malaria are not yet available. The chief objectives of this project are to test the hypotheses that specific point mutations in Plasmodium falciparum DHFR and DHPS account for in vivo resistance to PS, and that specific genotypes of cg2 account for in vivo resistance to chloroquine. Host factors that may contribute to drug failure will also be assessed. Molecular assays will be developed for use as tools for epidemiological mapping of drug-resistant malaria. The proposed research will also study the mechanisms by which drug pressure causes the spread of resistance-conferring mutations both in individual infections and in epidemiological populations. Finally, surveillance for new mutations and genotypes will be conducted, and new mutations will be assessed in transfection studies to determine their role in resistance. These studies are expected to provide tools for the surveillance and control of drug-resistant malaria and to yield information that will aid in designing new antimalarial drugs or drug combinations that will be less prone to the development of resistance. The tools and information arising from these studies will be shared with and disseminated to interested parties including recipients of related Multilateral Initiatives on Malaria grants and the NIAID Malaria Research and Reference Reagent Repository.